Ojima I. (ed.) Fluorine in Medicinal Chemistry and Chemical Biology

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Unique Properties of Fluorine and Their Relevance 21

of C – F · · · C = O contacts with the F · · · C distance below the sum of the van der Waals radii

and the C – F · · · C = O torsion angle toward 90 ° . A similar but intramolecular orthogonal

polar interaction between a CF

groups and a spatially close amide C = O group, which

plays a role in the folding of an extended indole molecule, has recently been reported

[111] .

Figure 1.15 illustrates an example of the polar interaction of a C – F bond with the

guanidinium carbon of the Arg residue in an enzyme, revealed by the X - ray crystal struc-

ture of atorvastatin - HMG - CoA reductase (HMG - CoA = 3 - hydroxy - 3 - methylglutaryl -

coenzyme A; the rate - determining enzyme in the biosynthesis of cholesterol). As mentioned

above (see section 1.1 ), atorvastatin is the best - selling cholesterol - lowering drug, which

binds to HMG - CoA reductase tightly (IC

= 8 nM). In the early stage of the drug discov-

ery, a series of phenylpyrrole - hydroxylactones were evaluated for their inhibitory activity

against HMG - CoA reductase, as shown in Figure 1.15 a. Then, the SAR study on the

4 - substituted phenyl analogues identiﬁ ed that the 4 - ﬂ uorophenyl analogue (R = F) was

the most active; that is, ﬂ uorine was better than hydrogen, hydroxyl or methoxy group

at this position [112] . This ﬁ nding eventually led to the discovery of atorvastatin. The

X - ray crystal structure of atorvastatin – HMG - CoA reductase [113] shows a strong

C – F · · · C(NH

)( = NH) polar interaction between the 4 - ﬂ uorophenyl moiety and the

Arg590 residue of the enzyme with a very short F · · · C distance (2.9 Å ), as illustrated in

Figure 1.15 b [101] .

Several other examples of similar polar interactions between a C – F bond in a ligand

and the guanidinium ion moiety of an Arg residue in protein have been found in PDB.

However, no linear C – F · · · H – N interactions are identiﬁ ed, reﬂ ecting the poor hydrogen -

bond accepting ability of a C – F bond. Instead of hydrogen bonding, a C – F bond is found

to orient either parallel or orthogonal to the plane of the guanidinium ion moiety, bearing

a delocalized positive charge. These examples conﬁ rm the ﬂ uorophilic character of the

guanidinyl side - chain of an Arg residue.

2.9 Å

OMe

(µM)

2.8

6.3

3.2

•1/2Ca

Arg590

(a) (b)

COO

–

Figure 1.15 (a) In vitro activity of HMG - CoA reductase inhibitors. (b) Orthogonal polar

interaction of the ﬂ uorine substituent of atorvastatin with Arg590 in HMG - CoA reductase.

22 Fluorine in Medicinal Chemistry and Chemical Biology

1.1.8 Isostere

A variety of natural and synthetic peptides exhibit biological activities, which can be

developed as pharmaceutical drugs or biochemical agents. However, those peptides con-

sisting of amide linkages are, in most cases, easily deactivated through cleavage of key

amide bonds by hydrolytic enzymes. Accordingly, it is very useful if a key amide bond is

replaced with noncleavable bond, keeping the characteristics of the amide functionality.

An amide linkage in a peptide such as 29a has an imidate - like zwitterioninc resonance

structure 29b . Since the contribution of this resonance structure is signiﬁ cant, free rotation

around the C – N bond is partially restricted because of the substantial double - bond char-

acter of the C – N bond (see Figure 1.16 ). Accordingly, it might be possible to replace an

amide linkage with its isostere (i.e., a molecule having the same number of atoms as well

as valence electrons [114] ), regarded as a “ peptide isostere. ”

The ﬁ rst and simplest attempt at the “ peptide isostere ” was made by replacing an amide

bond of enkephalin with a trans - oleﬁ n unit [115] , but it did not give a desirable effect.

However, computational analysis of a model amide, N - methylacetamide ( 30b ) and its iso-

steres, by semiempirical molecular orbital calculation revealed that ﬂ uorooleﬁ n 30c resem-

bled 30a much more closely than 30b [116] . To conﬁ rm this ﬁ nding based on a semiempirical

method, the ab initio analysis of 30a , ( E ) - 2 - butene ( 30b ), ( Z ) - 2 - ﬂ uoro - 2 - butene ( 30c ) and

( Z ) - 1,1,1 - triﬂ uoro - 2 - methyl - 2 - butene ( 30d ) was carried out using the Gaussian 03 program

(B3LYP/6 – 311++G * * ) [117] by one of the authors (T. Y.), which gave electrostatic poten-

tials of these compounds. The results are illustrated in Figure 1.17 .

As readily anticipated, 30a has a very negative oxygen, a highly positive carbonyl

carbon, a highly negative nitrogen, and a very positive NH hydrogen. In sharp contrast,

30b has a nonpolarized negative C = C bond and only weakly positive CH hydrogen. Thus,

it is apparent that 30b does not mimic 30a electronically. In contrast, 30c has an appro-

priately polarized C = C double bond, a negative ﬂ uorine atom in place of the oxygen atom

of amide 30a , and a positive CH in place of the NH moiety of amide 30a . Thus, 30b

indeed mimics 30a electronically. Finally, 30d has a nonpolarized C = C bond, a modestly

positive CH hydrogen, and three negative ﬂ uorine atoms. Thus, 30d mimics 30a electroni-

cally to some extent, although sterically the CF

group is much bulkier than an oxygen

atom.

••

29a 29b

30a

30b

30c

30d

Figure 1.16 Resonance structures of amide 29 , amide 30a , and its isosteres 30b – d .

Unique Properties of Fluorine and Their Relevance 23

30b30a

30d30c

Figure 1.17 Electrostatic potential of the model compounds 30a to 30d (color alteration

from red to blue describes the shift of the electronically rich to deﬁ cient circumstance). See

color plate 1.17.

A ﬂ uorooleﬁ n isostere was successfully introduced to a physiologically important

neuropeptide, Substance P ( 31 ) [118] . As Figure 1.18 shows, the ( E ) - ﬂ uoroethene isostere

replaced the peptide linkage between the Phe

- Gly

residues of 31 to give Substance P

analogue 32a and its epimer 32b [119] . The neurokinin - 1 receptor binding assay of 32a

disclosed that 32a was almost as potent as the original peptide 31 . On the other hand, its

epimer 32b was 10 times less potent than 32a , as anticipated.

Another ﬂ uorooleﬁ n isostere, ( E ) - triﬂ uoromethylethene, was also introduced to

antibiotic Gramicidin S ( 33a ), replacing the peptide linkage between Leu and Phe (two

sites) to give an analogue 33b , as illustrated in Figure 1.19 [120] . Variable - temperature

NMR measurements of 33a and 33b indicated that the NH(Leu) and NH(Val) protons were

forming intramolecular hydrogen bonds. In addition, NOESY experiments of 33a and 33b

showed interstrand NOEs between NH(Leu) and NH(Val) as well as NH(Leu) and H

(Orn)

in both compounds. Moreover, X - ray crystallographic analysis of 33c conﬁ rmed the pres-

ence of a pair of intramolecular hydrogen bonds between NH(Leu) and C = O(Val) (1.96 Å

and 2.00 Å ). However, the replacement of the Leu - Phe amide linkage with the bulky ( E ) -

triﬂ uoromethylethene isostere caused a 70 ° twist in the plane of the C = C double bond

24 Fluorine in Medicinal Chemistry and Chemical Biology

Leu-Met-NH

Arg-Pro-Lys-Pro-Gln-Gln-Phe

Leu-Met-NH

Arg-Pro-Lys-Pro-Gln-Gln-Phe

2 nM

20 nM

1.3 nM

31 Substance P

32a R

= Bn, R

= H

32b R

= H, R

= Bn

Figure 1.18 Substance P ( 31 ) and its ﬂ uorooleﬁ n dipeptide isosteres ( 32a, b ).

(CH

)

(CH

)

(CH

)

NHR

RHN(CH

)

33a

33b (R = H)

33c

(R = Cbz)

Figure 1.19 Gramicidin S ( 33a ) and its CF

- containing isostere ( 33b ).

compared with the amide moiety in the original β - turn structure of 33a . This is very likely

attributable to the bulkiness of the CF

groups, which are difﬁ cult to be accommodated

inside the β - turn hairpin structure. In spite of some conformational difference, 33b was

found to possess high antibiotic activity against Bacillus subtilis equivalent to that of 33a

[120] . The use of the ( E ) - triﬂ uoromethylethene peptide isostere as β - turn promoter

and peptide mimetics in general has also been studied [121] . A fairly large dipole moment

of this isostere ( µ = 2.3 D) appears to play a key role in the improved mimicry of the

electrostatic potential surface of the amide linkage (dipole moment of the amide unit:

µ = 3.6 D) [120] .

Although ﬂ uoroethene and triﬂ uoroethene peptide isosteres were successfully

employed as nonhydrolyzable amide substitutes, more recently triﬂ uoroethylamines have

emerged as highly promising peptide isosteres. The triﬂ uoroethylamine isostere has been

studied for partially modiﬁ ed retro - and retro - inverso Ψ [NHCH(CF

)]Gly peptides [122,

123] . Furthermore, it has been demonstrated that the CF

group of the triﬂ uoroethylamine

Unique Properties of Fluorine and Their Relevance 25

isostere can function as the carbonyl group of an amide to provide a metabolically stable,

nonbasic amine that has excellent hydrogen - bonding capability due to the strong inductive

effect of the CF

group [124, 125] . These unique characteristics of the triﬂ uoroethylamine

isostere have been exploited and quite successfully applied to the optimization of cathepsin

K inhibitors, which are promising antiresorptive agents for treatment of osteoporosis [124] .

As Figure 1.20 shows, triﬂ uoroethylamine analogue 34a exhibits 1000 time better potency

than the corresponding ethylamine analogue 34b . Pentaﬂ uoroethylamine analogue 34c

possesses a somewhat reduced activity, but still in a comparable level to 34a . Tosylmethyl-

amine analogue 34d shows virtually the same activity as that of 34c . The docking study

using the cathepsin K crystal structure has revealed that 34a forms a critical hydrogen

bond with the carbonyl oxygen of Gly66, as anticipated. The fact that the CF

group of

this isostere is attached to an sp

carbon provides ﬂ exibility in the orientation of the NH

bond to form hydrogen bonding in the most favorable manner [124] . Optimization of the

aromatic group of 34a led to the discovery of the exceptionally potent inhibitor 35a ,

exhibiting an IC

of 5 pM or less. The corresponding amide 35 is also extremely potent

(IC

= 0.015 nM) but is metabolically labile. Thus, 35a has been identiﬁ ed as the most

potent and promising drug candidate in this study [124] .

It has been shown that 2,3 - diﬂ uorotoluene ( 36 ) serves as a nonpolar shape mimic of

thymine ( 37 ) and diﬂ uorotoluene deoxyriboside ( 38 ) is, surprisingly, an excellent nonpolar

nucleoside isostere of thymidine ( 39 ) (see Fig. 1.21 ) [126, 127] . Thus, the nucleotide of

36 was incorporated into DNA by several high - ﬁ delity DNA polymerases [126, 128] ,

35a (~0.005 nM)

34a (R = CF

: 0.9 nM)

34b (R = CH

: 988 nM)

34c (R = CF

: 2.4 nM)

34d (R = p-CH

-C

: 2.5 nM)

35 (0.015 nM)

Figure 1.20 Cathepsin K inhibitors 34 and 35 , bearing triﬂ uoroethylamine isosteres. Numbers

in parentheses are IC

values.

3736

Figure 1.21 Diﬂ uorotoluene ( 36 ), diﬂ uorotoluene deoxyriboside ( 38 ), thymine ( 37 ), thymi-

dine ( 39 ) and adenine ( 40 ).

26 Fluorine in Medicinal Chemistry and Chemical Biology

forming a positional pair with adenine ( 40 ) in place of 37 without perturbing the double

helix structure, which was conﬁ rmed by X - ray crystallography (see Fig. 1.21 ) [127] . It

has also been shown that diﬂ uorotoluene does not form appreciable hydrogen bonds with

adenine, and hence it is nonselective in base pairing and destabilizing DNA in the absence

of DNA polymerases [128] . The probability of thymidine triphosphate choosing a wrong

partner is less than 0.1% and that of the triphosphate of 38 is less than 0.3% [126] . Thus,

the results clearly indicate that 36 is virtually a perfect shape mimic of 37 for DNA

polymerases.

Since these surprising ﬁ ndings, ﬁ rst made in 1997, challenge the Watson – Click base -

pairing principle in DNA (although the diﬂ uorotoluene isostere is only a minor part of

DNA), there has been a debate on the interpretation of the results, including the polarity

of 36 and possible F · · · HN as well as CH · · · N hydrogen bonding interactions [129] . However,

the X - ray crystallographic data [127] as well as extensive computational analysis [130]

unambiguously conﬁ rmed that 36 is indeed a nonpolar thymine isostere. Thus, these

ﬁ ndings have opened a new avenue of research on a variety of nonpolar nucleoside

isosteres [128] .

1.1.9 Diﬂ uoromethylene as Isopolar Mimic of the Oxygen Component of

P – O – C Linkage

Diﬂ uoromethylene has been recognized as an isopolar mimic of the oxygen component

of P – O – C or P – O – P linkage in phosphates, which can be used to generate nonhydrolyz-

able phosphate analogues of nucleotides, enzyme substrates, and enzyme inhibitors [131 –

137] . For example, a diﬂ uoromethylene linkage was successfully introduced to a

protein - tyrosine phosphatase (PTP) inhibitor of insulin receptor dephosphorylation [132]

(see Figure 1.22 ). A comparison of a hexamer peptide inhibitor bearing a phosphonometh-

ylphenylalanine (Pmp) residue ( 41a ) with that having a phosphonodiﬂ uoromethylphenyl-

alanine (F

Pmp) residue ( 41b ) revealed that 41b was 1000 times more potent than 41a ,

retaining high afﬁ nity for the SH2 domain of PTP [132] . The marked difference in potency

observed for 41a and 41b can be ascribed to the fact that the diﬂ uoromethylene group

increases the acidity of the phosphonic acid moiety and maintains appropriate polarity.

41a (X = CH

: 100 µM)

41b (X = CF

: 0.10 µM)

AcHN

O O

pKa

6.4 (X = O)

7.6 (X = CH

)

6.5 (X = CHF)

5.4 (X = CF

)

Figure 1.22 Nonhydrolyzable phosphate mimics 41 and their estimated pKa values. Numbers

within the parentheses are IC

values.

Unique Properties of Fluorine and Their Relevance 27

A similar incorporation of a P – CF

unit as a P – O bond surrogate to sphingomyelin

was reported [134] for the development of nonhydrolyzable inhibitors of sphingomyelin-

ase (SMase), which cleaves sphingomyelin to release ceramide. As Figure 1.23 shows,

diﬂ uoromethylene analogue 42b is twice as potent as methylene analogue 42a in the

inhibition of SMase from B. cereus [134] .

Norcarbovir triphosphate ( 43d ) is a potent inhibitor of HIV reverse transcriptase,

comparable to AZT and carvovir, but is amenable to enzymatic hydrolysis in vivo [135] .

Thus, methylenediphosphonate, ﬂ uoromethylenediphosphonate, and diﬂ uoromethylenedi-

phosphonate analogues of 43d were synthesized to block the enzymatic hydrolysis and

evaluated for their potency and stability in human fetal blood serum [135] . As Figure 1.23

shows, the methylenediphosphonate analogue ( 43a ) is inactive, and the ﬂ uoromethylene-

diphosphonate analogue ( 43b ) shows only a moderate potency. Diﬂ uoromethylenediphos-

phonate analogue 43c is the most active among these nonhydrolyzable analogues with IC

of 5.8 µ M, which is 10 times weaker than that of 43d . Nevertheless, 43c possesses > 40

times longer half - life (45 h) than the natural enantiomer of 43d (65 min) and > 500 times

more stable than AZT triphosphate (5 min) [135] .

The ﬂ uoromethylene and diﬂ uoromethylene linkages have also been incorporated

into an aspartyl phosphate, providing the ﬁ rst synthetic inhibitors of aspartate semialde-

hyde dehydrogenase [136] as well as lysophosphatidic acid analogues, which increased

the half - lives of analogues in cell culture [137] .

The use of a diﬂ uoromethylene unit as a surrogate of a carbonyl group is another

logical extension. In fact, diﬂ uoromethylene analogues of the inhibitors of the rotamase

activity of FK506 binding protein 12 (FKBP12), which catalyzes cis – trans isomerization

of a peptidyl - prolyl bond, have been investigated [138] . As Table 1.8 shows, the K

values

of diﬂ uoromethylene analogues, 44a and 44d , for the FKBP12 inhibition (rotamase activ-

ity) are comparable to or better than those of the carbonyl counterparts, 44b and 44e . It

should be noted that the corresponding simple methylene analogues, 44c and 44f do not

show any appreciable activity, which suggests that the gem - diﬂ uoromethylene group par-

ticipates in some speciﬁ c interactions with the protein. After optimization of the proline

and its ester moieties, the most potent inhibitor 45 ( K

19 nM) was developed. The X - ray

crystal structure of the 45 – FKBP12 complex strongly suggests that the two ﬂ uorine atoms

participate in the moderate - to - weak hydrogen bonding interactions with the Phe36 phenyl

ring as well as Tyr26 hydroxyl group.

42a (X = CH

: 120 µM)

42b (X = CF

: 57 µM)

NMe

O O O

HO HO

43a (Y = CH

: >100 µM)

43b (Y = CHF: 34.8 µM)

43c (Y = CF

: 5.8 µM)

43d (Y = O: 0.5 µM)

Figure 1.23 SMase inhibitors ( 42 ) and FKBP 12 rotamase inhibitors ( 43 ). Numbers within

the parentheses are IC

values.

28 Fluorine in Medicinal Chemistry and Chemical Biology

1.1.10 High Electrophilicity of Fluoroalkyl Carbonyl Moieties

Successive substitution of hydrogen atoms in acetone with ﬂ uorine atoms clearly lowers

the frontier orbital energy levels by 0.2 – 0.6 eV on the basis of ab initio computation (see

Table 1.9 ). It is worthy of note that the positive charge at the carbonyl carbon and the

negative charge at the oxygen atoms in these ketones, decrease as the number of ﬂ uorine

atoms increases. The validity of the computational analysis is conﬁ rmed experimentally

by the

C NMR chemical shifts of these carbons, which decrease from 206.58 ppm for

acetone to 172.83 ppm for hexaﬂ uoroacetone, in good agreement with the carbonyl carbon

charges, as shown in Table 1.9 .

The substantial decrease in the HOMO (highest occupied molecular orbital) energy

level of triﬂ uoroacetophenone ( 46b ), compared with acetophenone ( 46a ) was observed

[139] by

C NMR when a 1:1 mixture of 46a and 46b was treated with BF

· O E t

. Thus,

a 16.7 ppm downﬁ eld shift took place only for the carbonyl carbon atom of 46a , showing

the coordination of the carbonyl group to the Lewis acid, while no change in the

chemical shift was observed for 46b (see Scheme 1.3 ). This marked difference in reactivity

is attributed to the substantially decreased basicity of the carbonyl oxygen in 46b caused

by the strong inductive effect of the triﬂ uoromethyl moiety. This selectivity was demon-

strated in the highly chemoselective reduction of 46a and 46b under two different reaction

conditions, as illustrated in Scheme 1.3 . Thus, the reduction of a 1:1 mixture of 46a and

46b with tributyltin hydride gave 1 - phenyl - 2,2,2 - triﬂ uoroethanol ( 48b ) as the sole product

in 40% yield, while the same reaction in the presence of one equivalent of BF

· OEt

− 78 ° C afforded 1 - phenylethanol ( 48a ) exclusively in 82% yield.

Table 1.8 FKBP 12 rotamase inhibition [138]

CO OCH

OCH

CO OCH

OCH

44 45

Compound X Y

( µ M)

44a F

N 0.872

44b O N 4.00

44c H

–

44d F

CH 1.30

44e O CH 2.20

44f H

–

45 0.019

No appreciable inhibition at 10 µ M.

Unique Properties of Fluorine and Their Relevance 29

Table 1.9 Calculated HOMO and LUMO levels of ﬂ uorinated acetones

Compound Energy Level (eV) Charges

C NMR chemical

shift of C = O

HOMO LUMO C = O C = O

C(O)CH

− 7.054 − 0.784

0.573

− 0.553

206.58

FC(O)CH

− 7.507 − 1.247

0.544

− 0.547

205.62

CHF

C(O)CH

− 7.911 − 1.873

0.518

− 0.513

197.38

C(O)CH

− 8.278 − 2.098

0.505

− 0.486

189.33

C(O)CF

− 9.384 − 3.476

0.421

− 0.419

172.83

Computation was carried out by one of the authors (T.Y.) using the Gaussian 03W at the B3LYP/6 – 311++G * * level.

Ref. [132] .

Ref. [140] .

46a 46b

48a 48b

conditions

n-Bu

SnH (1.0 equiv)/

CN, rt, 5 h

n-Bu

SnH, BF

·OEt

(1.0 equiv)/

, –78 °C, 1 h

40%

82%

47a 46b

·OEt

Scheme 1.3 Reactions of 46a and 46b with BF

· OEt

and n - Bu

SnH.

1.1.11 Use of Diﬂ uoromethyl and Triﬂ uoromethyl Ketones as Transition State

Analogues in Enzyme Inhibition

Tetrahedral transition state analogues of ester and amide substrates are known to function

as efﬁ cient enzyme inhibitors of hydrolytic enzymes such as serine and aspartyl proteases

as well as metalloproteinases (see Fig. 1.24 ) [141 – 144] . Although ketals of alkyl or aryl

ketones are usually not stable, those of diﬂ uroalkyl or triﬂ uoromethyl ketones have con-

siderable stability, as exempliﬁ ed by their facile formations of the corresponding stable

hydrates [145, 146] . Therefore, substrate analogues, containing diﬂ uoroalkyl or triﬂ uoro-

methyl ketone moiety in appropriate positions, have been studied as effective transition

state inhibitors of hydrolytic enzymes [141, 147 – 150] .

30 Fluorine in Medicinal Chemistry and Chemical Biology

For example, the rather simple diﬂ uoroalkyl ketone 49c and triﬂ uoromethyl ketone

49b were designed as inhibitors of acetylcholinesterase (AchE), a serine esterase [151] .

As Figure 1.25 shows, these ﬂ uoroketones exhibit excellent AchE inhibitory activities with

nanomolar level K

values, and 49c possesses 200,000 times higher potency ( K

= 1.6 nM)

than the corresponding alkyl ketone 49a [151] . This remarkable result can be readily

rationalized by taking into account the strong electrophilicity of the carbonyl group of

diﬂ uoroalkyl and triﬂ uoromethyl ketones, described in the preceding section.

In a similar manner, a variety of transition state analogues bearing a triﬂ uoroacetyl

moiety as the key functional group have been designed and synthesized as the inhibitors

of human neutrophil elastase [152, 153] , human cytomegalovirus protease [154, 155] ,

human leukocyte elastase [156, 157] , and other enzymes. Some of these inhibitors exhibit

extremely high potency. For example, 51 has a K

of 3.7 pM against AchE [158] and 50

has an IC

of 0.88 nM against insect juvenile hormone esterase [159] (see Figure 1.26 ).

Diﬂ uoroalkyl ketones have also been successfully employed as the key structure of

renin inhibitors. Renin is an aspartyl protease and transforms angiotensinogen (452 amino

acid residues for human) to angiotensin I, which is further cleaved by angiotensin -

converting enzyme (ACE) to angiotensin II, which exerts a vasoconstricting effect [160] .

Accordingly, renin inhibitors have been extensively studied for their use in controlling

hypertension. Although human angiotensinogen has 452 amino acid residues, the ﬁ rst

dodecapeptide sequence is the most important for its activity (see Figure 1.27 ) [160] .

Enzyme-Ser-OH

Enzyme-Ser

(AA)

(AA')

Enzyme-Ser-OH

(AA)

(AA')

(AA)

(AA')

Enzyme-Ser

Enzyme-Ser-OH

OOH

F F

Tetrahedral transition state for hydrolysis

Stabel tetrahedral intermediate

Figure 1.24 Mechanism of peptide hydrolysis by a serine protease and enzyme inhibition

by forming stable tetrahedral intermediate.

49a (X = H, Y = H) 310,000

1.6

49b (X = F, Y = H)

49c (X = H, Y = F)

(nM)

Acetylcholine

Figure 1.25 Transition state inhibitors of AchE based on diﬂ uoroalkyl and triﬂ uoromethyl

ketone substrate analogues.